In the fields of molecular biology and microbiology, it has long been common to employ solid supports having biomolecules immobilized thereon. Immobilization provides various advantages, such as, allowing for multiplexing of samples and ready measurements of tags employed in a large number of signal systems. There is, for example, a wealth of literature about conjugating nucleic acid polymers to beads.
One such method uses streptavidin-coated magnetic beads, which are commercially available from, e.g. Dynal of Oslo, Norway. These beads are contacted with terminally labeled biotinylated oligonucleotides. Biotin and streptavidin are well known to interact in a non-covalent but highly stable manner, which for many purposes is the functional equivalent of covalent conjugation. Thus, oligonucleotides may become immobilized on a bead through this biotin-streptavidin interaction.
Another method relies on carbodiimide chemistry. There are several variations on this basic approach. For example, hydrazide-coated beads may be joined to carboxy-modified oligonucleotides as described in. e.g. Kremsky, J. N. et al. Nucleic Acids Res. 15:2891-2909, 1987, via carbodiimide-promoted coupling. Another variation on this approach uses controlled pore glass (CPG) or magnetic polystyrene beads with a surface coating of carboxyl groups, which are reacted with amino-modified oligonucleotides, as described in, e.g., Ghosh, S. S. et al. Nucleic Acids Res. 15:5353-5372, 1987 and Lund V. et al. Nucleic Acids Res. 16:10861-10880, 1987.
Yet another method uses a layer of polyethyleneimine (PEI) that surrounds a bead. See, e.g., Wasserman, B. P. et al. Biotech. and Bioeng. XXII:271-287, 1980; Povey, A. C. et al. J Pharmaceutical Sciences 75:831-837, 1986; Rounds, M. A. J. Chrom. 362:187-196, 1986; Van Ness, J. et al. Nucleic Acids Res. 19:3345-3350, 1991; and PCT International Publication WO 94/00600. Furthermore, PEI has been used to immobilize various biomolecules, including nucleic acid polymers, on a wide variety of non-bead solid supports and surfaces See, e.g., Schurer, J. W. et al. J. Histochemistry and Cytochemistry 25:384-387, 1977; Alpert, A. J. et al. J. Chrom. 185:375-392, 1979; Vanecek, G. et al. Anal. Biochem. 121:156-169, 1982; El Rassi, Z. et al. Chomatographia 19:9-18, 1984; Rounds, M. A. J. Chrom. 362:187-196, 1986; European Patent Application No. 0,197,784 A1 to Swann, W. E. et al., 1986; D'Souza, S. F. Biotechnology Lett. 8:643-648, 1986; Povey, A. C. et al. J. Pharmaceutical Sciences 76:201-207, 1987; U.S. Pat. No. 4,753,983 to Ngo, T. T., 1988; European Patent Application No. 0,403,700 A1 to Crane, L. J. et al, 1990; Trinh, C. K. Die Angewandte Makromol. Chemie 212:167-179, 1993; Bruil, A. et al J. Biomed, Mat. Res. 27:1253-1268, 1993; PCT International Publication No. WO 95/02184;. Indeed, PEI has been reported to bind oligonucleotides in the absence of a solid support. See, e.g., Boussif O. et al. Proc. Natl. Acad. Sci. USA 92:7297-7301, 1995.
Recently, intense attention has focused on creating arrays of biomolecules, and particularly polynucleotides, on a flat solid support. The following publications (and the references cited therein), which are exemplary only, provide general and specific overviews of various utilities for these biomolecular arrays, as well as methods of preparing such arrays: Eggers, M. D. et al. Advances in DNA Sequencing Technology SPIE Vol. 1891:113-126, 1993; Chetverin, A. B. et al. Bio/Technology 12:1093-1099, 1994; Southern, E. M. Nucleic Acids Research 22:1368-1373, 1994; Lipshutz, R. J. et al. BioTechniques 19:442-447, 1995; Schena, M. BioEssays 18:427-431, 1996; Blanchard, A. P. et al. Biosensors & Bioelectronics 11:687-690, 1996; O'Donnell-Maloney, M. J. et al. Genetic Analysis: Biomolecular Engineering 13:151-157, 1996; Regalado, A. Start-Up 24-30, Oct. 1996; and Stipp, D. Fortune pp. 30-41, Mar. 31, 1997.
Many of the reported procedures for preparing these arrays has drawn upon expertise which developed in connection with immobilizing biomolecules to beads and other solid supports. Perhaps surprisingly, there has been, to date, no report of using PEI to immobilize biomolecules in an array format. At most, this has been suggested as a theoretical possibility for linking oligonucleotides to a nylon support. See, e.g., PCT International Publication No. WO 95/09248, citing Van Ness. J. et al. Nucleic Acids Res. 19:3345-3350, 1991.
As disclosed herein, the present inventors have identified problems associated with preparing PEI-based oligonucleotide arrays, and have solved those problems, so that PEI-based arrays are now more than just a theoretical possibility. Furthermore, the PEI-based arrays of the invention provide various advantages compared to other types of arrays, as also disclosed herein.